Structured illumination microscope apparatus and an image forming apparatus

ABSTRACT

Polarized light which is emitted from an optical fiber becomes circular polarized light bypassing through a first quarter wave plate. The circular polarized light which has entered a second quarter wave plate is converted into nearly linear polarized light which has S polarization. P polarization components are removed from the nearly linear polarized light by a polarizer, but the polarizer is not always necessary. The optical axis of the polarizer is set to be a direction which allows transmitting of S polarized light. The light that has passed through the polarizer is separated into diffracted lights by a diffraction grating, and is used as the structured illumination light.

BACKGROUND

The present invention relates to a structured illumination microscopedevice and an image forming apparatus.

In the field of observation and measurement of the micro-structures ofsamples, observation at higher spatial resolution is demanded. As amethod to improve the lateral resolution of a sample, a method ofmodulating a sample with structured lights during photographing anddemodulating the captured image by image processing is known as“structured illumination microscopy”, and is disclosed in U.S. Pat. No.6,239,909 and US RE38,307, for example.

The sixth embodiment disclosed in U.S. Pat. No. 6,239,909 is an examplewhen the structured illumination microscope apparatus is applied to afluorescence microscope, and the optical system thereof splits theillumination light emitted from a coherent light source using such beamsplitting means as a diffraction grating, then condenses theillumination beams into a pupil plane of an objective lens, and emitsthe illumination beams as parallel beams from the objective lens atdifferent angles, so as to overlap around the observation object andform an interference fringe.

Because the illumination light is modulated into a fringe, diffractedlights containing spatial frequency components of the shape informationof the observation object, which cannot be transferred by a conventionalimaging system, can be used for image formation. And by relativelymodulating the phase of the split illumination beams and moving theinterference fringe on the observation object when a plurality of imagesare acquired, image formation based on image computing processing isenabled.

In concrete terms, phase modulation is performed by moving thediffraction grating vertically to the optical axis, or in anotherexample, by inserting a wedge prism into one of the illumination paths,and moving this wedge prism in a direction vertical to the optical axis.

According to the method disclosed in US RE38,307, illumination lightfrom the coherent light source is guided using an optical fiber, and issplit by such beam splitting means as a diffraction grating, and thenthe illumination beams are condensed into a pupil plane of the objectivelens so as to form an interference fringe near the observation object.Because the illumination light is modulated into a fringe, the highfrequency components of the shape information of the observation object,which cannot be transferred by a conventional imaging system, can beused for image formation. And a plurality of images are captured in thesame manner, and image formation based on image computing processing isperformed.

According to this method, in order to create one image, not only aplurality of images are acquired with phase-modulated structuredillumination, but also images are acquired with changing the directionof the structured illumination. This is because the high frequencycomponents can be used for image formation only when the structure hasthe same direction as the direction of the structured illumination, soin order to restore the shape of the sample which extendstwo-dimensionally, a plurality of images must be acquired with changingthe direction of the structured illumination, and be combined.

In this structured illumination, it is normally desirable that the beamswith respect to the interference surface have S polarization in order toallow two or more beams to interfere. Because the contrast of theinterference fringe when beams with S polarization is 1, regardless ofthe incident angle, the contrast when beams enter in the P polarizationstate attenuates in proportion to cos (Δθ), where Δθ is the crossingangles of the beams. Since the observed light is the sum of Spolarization and P polarization, contrast to be observed decreases as Ppolarization attenuates. At Δθ>90°, the numeric value of the contrast ofP polarization becomes negative, which means that thebrightness/darkness of the interference fringe inverts, and theinterference fringe generated by S polarization is cancelled, which isnot desirable.

In particular, the structured illumination microscope is a technologyused to obtain high resolution, and it is preferable that the NA of theobjective lens to be used is as high as possible, and the pitch of thestructured illumination is as short as possible. As a result, the beamsfor the structured illumination enter into the sample at a large angle,so if a P polarization component exists, attenuation of the Ppolarization is major since the above mentioned Δθ is large, whichbecomes the cause of deterioration of the structured illuminationcontrast.

In order to address this problem, a non-polarization state is created byallowing light from the light source to pass through a diffuser orvibrating multi-mode optical fibers, then light is nearly linearpolarized using a polarizer, which is installed near the diffractiongrating and which rotates coaxially with the diffraction grating, so asto enter with S polarization onto the sample.

As a result, ideal structured illumination contrast is obtained, but thepolarizer blocks about half of the light power, which lowers utilizationefficiency of the light to 50%.

SUMMARY

In view of the foregoing, it is an object of the present invention toprovide a structured illumination microscope of which utilizationefficiency of the light from the light source is high.

It is another object of the present invention to provide an imageforming apparatus of which utilization efficiency of the light from thelight source is high.

A first aspect of the present invention is a structured illuminationmicroscope apparatus comprising: an illumination optical system whichincludes a beam splitter rotatably disposed near a conjugate position ofa sample and splitting a beam from a light source into a plurality ofbeams, and which irradiates illumination light that has been spatiallymodulated by allowing the plurality of beams to interfere near a samplesurface; an imaging optical system which forms an image of diffractedlight generated from the sample on an image capturing surface of animage capturing device; an image processing device which generates asample image by processing a plurality of images captured by the imagecapturing device each time a phase of the spatially modulatedillumination light is changed; a first quarter wave plate which isdisposed between the light source and the beam splitter, and of whichdirection of a fast axis is relatively fixed with respect to apolarization direction of the light from the light source; and a secondquarter wave plate which is disposed between the first quarter waveplate and the beam splitter, and is rotatable coaxially with therotation axis of the beam splitter with the same rotation angle as thatof the beam splitter.

A second aspect of the present invention is a structured illuminationmicroscope apparatus comprising: an illumination optical system whichincludes a beam splitter disposed near a conjugate position of a sample,and splitting a beam from a light source into a plurality of groups ofbeams, of which splitting directions are different, around an opticalaxis; and a beam selector for selecting and transmitting only onearbitrary group of beams out of the plurality of groups of beams, andwhich irradiates illumination light that has been spatially modulated byallowing the plurality of beams included in the one group of beams tointerfere near a sample surface; an imaging optical system which formsan image of diffracted light generated from the sample on an imagecapturing surface of an image capturing device; an image processingdevice which generates a sample image by processing a plurality ofimages captured by the image capturing device each time a phase of thespatially modulated illumination light is changed; a first quarter waveplate which is disposed between the light source and the beam splitter,and of which direction of a fast axis is relatively fixed with respectto a polarization direction of the light from the light source; and asecond quarter wave plate which is disposed between the beam splitterand the beam selector, and of which rotation angle from the optical axisis relatively fixed with respect to the beam selector.

A third aspect of the present invention is an image forming apparatuscomprising, light source means for producing linearly polarized light,light splitting means, placed in a position that is optically conjugateto a specimen for splitting light from the light source means, aperiodic structured light means for providing a periodic structuredlight on a specimen by interference of the split light, shifting meansfor shifting the phase of the periodic structured light, producing meansfor producing images of light from the specimen onto an image plane,detecting means for detecting and recording the images of the specimenin the image plane, and processing means for producing a reconstructionof the specimen by processing the recorded images which are taken withdifferent phase of the periodic structured light, wherein a firstquarter wave retarding means is located between the light source meansand the light splitting means, the first quarter wave retarding means ismounted in a fixed orientation relative to the polarization of the lightsource, to produce circularly polarized light, and a second quarter waveretarding means is located between the first quarter wave retardingmeans and the light splitting means, the second quarter wave retardingmeans is enabled to co-rotate with the light splitting means.

A fourth aspect of the present invention is an image forming apparatuscomprising, light source means for producing linearly polarized light,light splitting means placed in a position that is optically conjugateto a specimen, for splitting the light from the light source means intoa plurality of beams of light, light selecting means to select a subsetof beams from among the plurality of beams of light, a periodicstructured light means for providing a periodic structured light on thespecimen by interference of the split light, producing means forproducing images of light from the specimen onto an image plane,detecting means for detecting and recording the images of the specimenin the image plane, and processing means for producing a reconstructionof the specimen by processing the recorded images which are taken withdifferent phase of the periodic structured light, wherein a firstquarter wave retarding means is located between the light source meansand the light splitting means, the first quarter wave retarding means ismounted in a fixed orientation relative to the polarization of the lightsource, to produce circularly polarized light, and a second quarter waveretarding means is located between the first quarter wave retardingmeans and the specimen, the second quarter wave retarding means isenabled to co-rotate with the light selecting means.

A fifth aspect of the present invention is an illumination opticalsystem which includes a beam splitter rotatably disposed near aconjugate position of a sample and splitting a beam from a light sourceinto a plurality of beams, and which irradiates illumination light thathas been spatially modulated by allowing the plurality of beams tointerfere near a sample surface, comprising: a first quarter wave platewhich is disposed between the light source and the beam splitter, and ofwhich direction of a fast axis is relatively fixed with respect to apolarization direction of the light from the light source; and a secondquarter wave plate which is disposed between the first quarter waveplate and the beam splitter, and is rotatable coaxially with therotation axis of the beam splitter with the same rotation angle as thatof the beam splitter.

A sixth aspect of the present invention is an illumination opticalsystem which includes a beam splitter disposed near a conjugate positionof a sample, and splitting a beam from a light source into a pluralityof groups of beams, of which splitting directions are different, aroundan optical axis; and a beam selector for selecting and transmitting onlyone arbitrary group of beams out of the plurality of groups of beams,and which irradiates illumination light that has been spatiallymodulated by allowing the plurality of beams included in the one groupof beams to interfere near a sample surface, comprising: a first quarterwave plate which is disposed between the light source and the beamsplitter, and of which direction of a fast axis is relatively fixed withrespect to a polarization direction of the light from the light source;and a second quarter wave plate which is disposed between the beamsplitter and the beam selector, and of which rotation angle from theoptical axis is relatively fixed with respect to the beam selector.

Further applicability of the present invention will become apparent fromthe detailed description given hereinafter. However, it should beunderstood that the detailed description and specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below and the accompanying drawingswhich are given by way of illustration only and thus are not limiting ofthe present invention.

FIG. 1 is a diagram depicting an overview of the optical system of astructured illumination microscope apparatus according to a firstembodiment of the present invention;

FIG. 2 is a diagram depicting a direction of an axis of each opticalelement and a polarization state of beams according to the firstembodiment;

FIG. 3 is a graph showing the relationship of the phase error of thefirst quarter wave plate, the phase error of the second quarter waveplate and the drop in illumination intensity of nearly linear polarizedlight;

FIG. 4 is a graph showing the relationship of the phase error of thefirst quarter wave plate, the phase error of the second quarter waveplate, and the first structured illumination direction, which minimizesthe drop in illumination intensity of nearly linear polarized light;

FIG. 5 is a graph showing the characteristics of an example of awideband wave plate (achromatic wave plate);

FIG. 6 is a graph depicting an example of the characteristics of twoquarter wave plates having different design waves;

FIG. 7 is a diagram depicting an optical system of a structuredillumination microscope apparatus according to a second embodiment ofthe present invention;

FIG. 8 are diagrams depicting a diffraction grating and condensingpositions of the diffracted light according to the second embodiment ofthe present invention;

FIG. 9 is a diagram depicting an overview of a beam selector accordingto the second embodiment of the present invention;

FIG. 10 is a diagram depicting a direction of an axis of each opticalelement and polarization state of beams according to the secondembodiment of the present invention; and

FIG. 11 are diagrams depicting a method for phase modulation accordingto the second embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Examples implementing the present invention will now be described withreference to the drawings, but this description does not limit the scopeof the present invention. FIG. 1 is a diagram depicting an overview ofan optical system of a structured illumination microscope apparatusaccording to a first embodiment of the present invention.

A coherent light from a light source LS is guided via an optical fiber1, and is converted into parallel lights by a collector lens 2. Theillumination light from the emitting end of the optical fiber 1 passesthrough a quarter wave plate 51 (first quarter wave plate), which issecured such that a fast axis is +45° inclined from the polarizationdirection thereof. Here the axis with least phase lag of the quarterwave plate refers to a direction of polarization when, the phase lagbecomes the least when a polarized light passes through the quarter waveplate. As a result, the illumination light becomes circular polarized ingeneral. The inclination of the fast axis need not accurately be +45°,but may have about a ±5.7° margin of error.

This tolerance corresponds to a 1% intensity variance.

The circular polarized light passes through a quarter wave plate 52(second quarter wave plate), which is secured in a diffraction grating 3and can rotate with the diffraction grating 3, and becomes nearly linearpolarized light. If the direction of the fast axis of the quarter waveplate 52 is set to be −45° from the ruling direction of the diffractiongrating 3, then the circular polarized light can be converted intonearly linear polarized light with which S polarization for the samplesurface is generated with respect to the diffraction direction of thediffraction grating. The direction of the fast axis need not be exactly+45°, but may have about a ±5.7° margin of error.

In this way, a roughly perfect S polarization for the sample surface canbe implemented, but if the remaining P polarization for the samplesurface is further removed, a polarizer 53, which has a transmissionpolarization direction that is in parallel with the ruling direction ofthe diffraction grating 3, may be disposed.

For the light source, a laser is used since the illumination light mustbe coherent. Therefore the polarization state at the emission end of theoptical fiber 1 is roughly a linear polarization unless scrambling isperformed, but it is preferable to pass this light through the polarizer53 so as to remove the orthogonal components, which slightly remain dueto the angle error and phase error of the quarter wave plates 51 and 52.In the present example, a single mode fiber, maintaining thepolarization plane, is used for the optical fiber 1.

The diffraction grating 3 is a beam splitting means. For the beamsplitting means, other optical elements, such as a prism, can be usedunless the elements do not interrupt the coherency. In the presentexample, the direction of the diffracted light can be changed byrotating the diffraction grating 3. A lens 4 and a lens 7 are set sothat the image of the diffraction grating 3 is formed on a conjugateplane of the sample, and a field stop 8 is disposed at a position wherethe image of the diffraction grating 3 is formed. A conjugate plane of apupil of an objective lens 12 is formed by the lens 4 so as to makedistinct light spots for each order of diffraction, and a phase plate 5and beam block 6 are disposed near the pupil conjugate plane. Thediffraction grating 3 has a one-dimensional periodic structure in avertical direction to the sheet of FIG. 1. The periodic structure may becreated by density (transmittance) or by step difference (phasedifference), but phase difference is preferable because diffractionefficiency of ±1-order beams is high.

In the present example, only ±1st order beams are used for theillumination light, and 0th order beam and 2nd or higher order beams arecut by the beam block 6. Therefore the 0th order beam indicated by thesolid line does not actually exist after the beam block 6, but in FIG.1, only the optical axis is shown to make it easier to understand raytrace.

The phase plate 5 disposed near the conjugate plane of the pupil of theobjective lens 12 has a thickness of a transparent substrate, or has athin film with controlled thickness deposited, so that an appropriatephase lag is generated in one or both of the incident beams (±1st orderbeam). Thereby a phase difference is generated between +1st order beamand −1st order beam.

The lights that have passed through the field stop 8 are converted intoparallel lights by a field lens 9, and condensed on the pupil plane P ofthe objective lens 12 for each order of diffraction, via an excitationfilter 10 and a dichroic mirror 11, and thus each beam from theobjective lens 12 becomes a parallel beam, irradiating onto the surfaceof the sample 13. Because of mutual coherent beams, the surface of thesample 13 is illuminated with an interference fringe pattern. Thisillumination light having a fringe structure is called a “structuredlight”, and this illumination method is called a “structuredillumination”.

If the sample 13 is illuminated by the structured illumination light,the illumination pattern and the structure of the sample 13 generate amoiré fringe, but this moiré fringe containing high frequencyinformation of the sample is a still lower frequency than the originalfrequency, so the moiré fringe can be imaged by the objective lens 12.

The lights (fluorescent) from the sample 13 are converted into parallellights by the objective lens 12, passed through the dichroic mirror 11and a barrier filter 14, and form a sample image on an image capturingsurface 16 of image capturing means 21 such as a CCD camera by a tubelens 15. This sample image is captured by an image storage and computingdevice 22.

As mentioned above, the captured images are the result after the sampleare modulated by structured illumination. So the sample image can beobtained first by processing the captured images using a known imagecomputing means of the image storage and computing device 22, next bydemodulating, and then by restoring the image, after which an ultra highresolution image of the sample can be displayed on the image displaydevice 23.

When the original image is restored by image processing, images arecaptured with modulating the phase of the structured illumination threeor more times for the same sample. This is because three unknownparameters of the 0th order component, the +1st order component and the−1st order component, out of the information on the frequency componentsof the sample diffracted by the structured illumination, exist in themodulated image and information more than the unknown quantity isrequired in order to determine the unknown quantity in computing.

Such imaging, computing/restoration and visualizing technologies areknown to be incorporated in conventional structured illuminationmicroscope apparatus, and are not directly related to the presentinvention, so a detailed description thereof is omitted.

In the case of the present example, phase modulation is performed byrotating the phase plate 5, but phase modulation may be performed bytranslationally driving the diffraction grating 3 in a direction togenerate diffraction. In this case the phase plate 5 is unnecessary.

FIG. 2 is a diagram depicting a direction of an axis of each opticalelement of this example and the polarization state of the beam. In FIG.2, a circle indicates a beam, and an arrow therein indicates a directionof polarization. A square indicates an optical element, and an arrowtherein indicates an axis direction.

Polarized light which is from the optical fiber 1 passes through thefirst quarter wave plate 51, and becomes a circular polarized light. Afast axis of the quarter wave plate 51, indicated in FIG. 2, refers to adirection of polarization in which phase lag is shortest when polarizedlight passes through the quarter wave plate. This is the same for thefast axis of the quarter wave plate 52 indicated in FIG. 2.

The relationship of the direction of polarization when the light is fromthe optical fiber 1 and the direction of the fast axis of the firstquarter wave plate 51 is that the fast axis of the first quarter waveplate 51 is +45° inclined from the direction of polarization of thelight from the optical fiber 1, as mentioned above.

The circular polarized light entered from the second quarter wave plate52 is converted into a nearly linear polarized light which has Spolarization for the sample surface. As mentioned above, the directionof the fast axis of the quarter wave plate 52 is −45° from the patterndirection of the diffraction grating 3.

The axis of the polarizer 53 is in a direction to transmit S polarizedlight for the sample surface. In the nearly linear polarized light,slightly remaining orthogonal components are removed by the polarizer53, but the polarizer 53 is not always necessary. The light that haspassed through the polarizer 53 is separated into diffracted lights bythe diffraction grating 3.

It is preferable to use the quarter wave plate 51 and quarter wave plate52 matching the wavelength of the light source, since the phasedifference is shifted from the original quarter wave if the wavelengthof the light source changes, but a wideband wave plate of which phasevariance with respect to the quarter wave is small in wide wavelengthsmay be used. In this case as well, the phase difference of an ordinarybeam and extraordinary beam should be within 90°±5° (quarter wave ± 1/72wavelength). The reason for this will be described with reference toFIG. 3.

In the graph of FIG. 3, the abscissa (unit: degrees) is a differencefrom the 90° phase difference (quarter wave) generated by the quarterwave plate 52, and a change of light intensity, when nearly linearpolarized light is obtained, is plotted, with respect to the differencesfrom the 90° phase difference generated by the quarter wave plate 51.The lines in FIG. 3 show the difference (unit: degrees) from the 90°phase difference generated by the quarter wave plate 51.

As FIG. 3 shows, as the difference from 90° (quarter length) increases,light intensity decreases in both the quarter wave plates, but if thephase error is kept within 5° in both quarter wave plates, the change oflight intensity when nearly linear polarized light is obtained can bewithin 1%. This means that in order to keep the change of lightintensity when nearly linear polarized light is obtained within 1%, thephase errors between the ordinary beam and extraordinary beam afterpassing through the phase plate should be within ±5% in both of thephase plates.

However the smaller the change in light intensity when the nearly linearpolarized light is obtained is, the better. Therefore the relationshipbetween the phases of the two quarter wave plates, which minimizes thechange in light intensity when nearly linear polarized light isobtained, was determined by simulation. The result is now described withreference to FIG. 4. In FIG. 4, the abscissa indicates a first directionθ1 (unit: degrees) of the structured illumination (the first directionout of many directions in which structured illumination is performedgenerally in the structured illumination microscope apparatus), and eachline in FIG. 4 corresponds to the difference (deviation) φ1 (unit:degrees) from the 90° phase difference of the first quarter wave plate51. The ordinate indicates the difference (deviation) φ2 (unit: degrees)from the 90° phase difference of the second quarter wave plate 53.

Each line shows the locus of the relationship between each parameterwhich minimizes the drop in the illumination light intensity when thenearly linear polarized light is obtained, while changing eachparameter. For example, if φ1=10° when the first structured illuminationdirection θ1 is 45°, then the change of the light intensity when thenearly linear polarized light is obtained can be minimized if φ2=−2.5°.In the same manner, if φ1=6° when the first structured illuminationdirection θ1 is 35°, then the change of light intensity when the nearlylinear polarized light is obtained can be minimized if φ2=−0.07°.

The relational expression of θ1, φ1 and φ2 created from this graph usingthe recurrence formula is as follows.

φ2=0.25×φ1×sin (6×θ1)

If some deviation from the optimum value is allowed, the relationship ofθ1, φ1 and φ2 can be determined such that the following expression (1)is established.

0.20×φ1×sin (6×θ1)≦φ2≦0.30×φ1×sin (θ1)  (1)

Then the change of light intensity when the nearly linear polarizedlight is obtained can be controlled to be within 0.5%.

In order to establish the relationship between φ1 and φ2 in expression(1), a wideband wave plate should be used. FIG. 5 shows an example ofthe characteristics of a wideband wave plate (achromatic wave plate).The abscissa indicates the wavelength (unit: nm) and the ordinateindicates the phase difference. As FIG. 5 shows, in the achromatic waveplate, the inclination of the phase difference with respect to thewavelength is generally sharper at the shorter wavelength side, andmilder at the longer wavelength side, compared with the wavelength atwhich the phase difference is the maximum.

Therefore as FIG. 6 shows, a relationship close to Expression (1) can beeasily established in the 460 nm to 560 nm wavelength range, if twoquarter wave plates having different design wavelengths are manufactured(units of the ordinate and abscissa in FIG. 6 are the same as those inFIG. 5), and the wave plate 1 (characteristics of the dashed line) isused as the wave plate 51 and the wave plate 2 (characteristics of thesolid line) is used as the wave plate 52. As a result, if wideband waveplates that satisfy a relationship close to expression (1) aremanufactured and used in combination, attenuation of the brightness ofthe structured illumination light can be prevented in a wide wavelengthband.

FIG. 7 is a diagram depicting an overview of an optical system of astructured illumination microscope apparatus according to a secondembodiment of the present invention. The only differences between thesecond embodiment and the first embodiment are the structure of thediffraction gratings 3 and 103, the positions of the quarter wave plates52 and 152, and the use of a beam selector 106. Other elements that arethe same as the elements of FIG. 1 are denoted with the same referencesymbols, and description on portions having the same functions may beomitted.

The second embodiment uses a diffraction grating 103 having a structureshown in FIG. 8A, instead of the diffraction grating 3 of the firstembodiment. This diffraction grating 103 has a periodic structure inthree directions with 60° intervals, and has a concave and convextriangular prisms arranged alternately. If a white triangle in FIG. 8Ais a concave portion, then a black triangle is a convex portion. Theapex angle of the triangles need not be exactly 60°, but 60° ispreferable.

FIG. 8B shows a state when the 0-order light and ±1-order lightsgenerated by the diffraction grating 103 are condensed at the conjugatepositions of the pupil via the lens 4 in FIG. 7. 110 in FIG. 8B showsthe 0th order's spot, and 111 a to 111 f are ±1st order's spot.

In the second embodiment, a beam selector 106 having a structure shownin FIG. 9 is used instead of the above mentioned beam block 6 of thefirst embodiment. The beam selector 106 has a mechanism whereby the beamselector 106 rotates with its center 106 a as the center of rotation.Areas 106 b and 106 c are holes or transparent with a glass of hightransmittance. Each time the beam selector 106 rotates, only a pair ofbeams, out of the pairs of beams 111 a and 111 d, 111 b and 111 e, and111 c and 111 f shown in FIG. 8B pass through the areas 106 b and 106 crespectively, and beams other than this pair are blocked by theshielding unit. The beam selector 106 is rotated by a rotation stage 142shown in FIG. 7, by which one out of the three sets of ±1st order beamsgenerated by the diffraction grating 103 is selected and allowed to passthrough at a time so as to be used for the structured illumination. Thebehavior of the selected two beams after the lens 7 are the same as thefirst embodiment. The 0th order diffracted light is blocked by the beamselector 106, and thereafter does not exist, but is still shown toclearly indicate the course of the beam, which is the same as FIG. 1.

If a timing of the areas 106 b and 106 c selecting the beams, and atiming of the imaging means capturing an image, are synchronized, thebeam selector 106 can rotate continuously at a predetermined speed. Forexample, as shown in FIG. 9, the shape of the edge of the beam selectoris matched with the areas 106 b and 106 c, and the timing of detectingthe edge is measured by a timing sensor. In the case of the firstembodiment, the diffraction grating 3 is rotated for a predeterminedangle, and is then stopped to capture the image, so it may take time forsettling, but in the second embodiment, an image can be captured athigh-speed since continuous rotation is possible.

Now polarization control of the second embodiment will be described withreference to FIG. 7 and FIG. 10. FIG. 10 is a diagram corresponding toFIG. 2 of the first embodiment, and shows the direction of the axis ofeach optical element and polarization state of the beam.

If the illumination light emitted from the end of the optical fiber 1passes through the quarter wave plate 151, which is secured with thefast axis thereof inclined at +45° from the polarization direction ofthe illumination light, the illumination light becomes roughly acircular polarized light. In the present example, the illumination lightin the circular polarized state enters the diffraction grating 103. Thenthe illumination light receives the diffraction function in the circularpolarized state. The second quarter wave plate 152 is disposed with thefast axis thereof inclined −45′ from the polarization direction in whichthe selected two beams become an S polarization for the sample surface,so that the second quarter wave plate 152 rotates along with the abovementioned beam selector 106. Thereby the illumination light can beconverted into a nearly linear polarized light in which the selected twobeams always become S polarization for the sample surface.

The phase modulation of the second embodiment is performed by theactuator 141 in FIG. 7. As FIG. 11A shows, the actuator 141translationally drives the diffraction grating 103 along one axis (xdirection in FIG. 11A). FIG. 11B is an enlarged view of the grating linedirection and driving direction. When a pitch of the grating is p, andthe angles of the grating lines with respect to the driving directionare θ1, θ2 and θ3 respectively, the converted pitches p1, p2 and p3 inthe driving direction become

p1=p/cos θ1

p2=p/cos θ2

p3=p/cos θ3

respectively. Therefore, for the phase modulation required for imageprocessing, the diffraction grating is driven with ⅓ of the convertedpitch (or a multiple thereof) at a time in the respective directions.

In the second embodiment, a polarizer is not used, but a polarizer maybe added just like the first embodiment, in order to Improve the finallinear polarization degree. In this case, the polarizer is disposedafter the second quarter wave plate 152 and before or after the beamselector 106 so as to be rotated together with the beam selector 106, ordisposed immediately after the emission end of the optical fiber 1. Thepolarization direction of the polarizer is a direction in which the twobeams become the S polarization state in the former case, and adirection in parallel with the polarizing direction of the optical fiber1 in the latter case.

As described above, the polarizer is not used for creating thestructured illumination light in the present invention, unless it isused as an option for removing the P polarization as noise, so almostall lights from the light source can be used for the structuredillumination light in the S polarization state, and the utilizationefficiency of the light can be increased.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention.

1. A structured illumination microscope apparatus comprising: anillumination optical system which includes a beam splitter rotatablydisposed near a conjugate position of a sample and splitting a beam froma light source into a plurality of beams, and which irradiatesillumination light that has been spatially modulated by allowing theplurality of beams to interfere near a sample surface; an imagingoptical system which forms an image of diffracted light generated fromthe sample on an image capturing surface of an image capturing device;an image processing device which generates a sample image by processinga plurality of images captured by the image capturing device each time aphase of the spatially modulated illumination light is changed; a firstquarter wave plate which is disposed between the light source and thebeam splitter, and of which direction of a fast axis is relatively fixedwith respect to a polarization direction of the light from the lightsource; and a second quarter wave plate which is disposed between thefirst quarter wave plate and the beam splitter, and is rotatablecoaxially with the rotation axis of the beam splitter with the samerotation angle as that of the beam splitter.
 2. A structuredillumination microscope apparatus comprising: an illumination opticalsystem which includes a beam splitter disposed near a conjugate positionof a sample, and splitting a beam from a light source into a pluralityof groups of beams, of which splitting directions are different, aroundan optical axis; and a beam selector for selecting and transmitting onlyone arbitrary group of beams out of the plurality of groups of beams,and which irradiates illumination light that has been spatiallymodulated by allowing the plurality of beams included in the one groupof beams to interfere near a sample surface; an imaging optical systemwhich forms an image of diffracted light generated from the sample on animage capturing surface of an image capturing device; an imageprocessing device which generates a sample image by processing aplurality of images captured by the image capturing device each time aphase of the spatially modulated illumination light is changed; a firstquarter wave plate which is disposed between the light source and thebeam splitter, and of which direction of a fast axis is relatively fixedwith respect to a polarization direction of the light from the lightsource; and a second quarter wave plate which is disposed between thebeam splitter and the beam selector, and of which rotation angle fromthe optical axis is relatively fixed with respect to the beam selector.3. The structured illumination microscope apparatus according to claim1, wherein the direction of the fast axis of the first quarter waveplate is +45°±5.7° with respect to the polarization direction ofincoming illumination light.
 4. The structured illumination microscopeapparatus according to claim 1, wherein the direction of a fast axis ofthe second quarter wave plate is −45°±5.7° with respect to a rulingdirection of the beam splitter.
 5. The structured illuminationmicroscope apparatus according to claim 1, further comprising apolarizer having a transmission polarization direction which is parallelwith a ruling direction of the beam splitter, immediately after thesecond quarter wave plate.
 6. The structured illumination microscopeapparatus according to claim 2, further comprising a polarizer which canrotate around the optical axis, which is disposed after the secondquarter wave plate and before or after the beam selector, and of whichrotation angle with respect to the beam selector and the optical axis isrelatively fixed.
 7. The structured illumination microscope apparatusaccording to claim 1, wherein the phase difference between an ordinarybeam and an extraordinary beam after passing through the first quarterwave plate and the phase difference between an ordinary beam and anextraordinary beam after passing through the second quarter wave plateare within 90°±5°, respectively.
 8. The structured illuminationmicroscope apparatus according to claim 1, wherein the relationship of0.20φ1×sin (6θ1)≦φ2≦0.30φ1×sin (6θ1) is established, where θ1 is a firststructured illumination direction, φ1 is a difference of the phasedifference generated by the first quarter wave plate from 90° (quarterwave), and φ2 is a difference of the phase difference generated by thesecond quarter wave plate from 90° (quarter wave).
 9. The structuredillumination microscope apparatus according to claim 2, wherein thedirection of the fast axis of the first quarter wave plate is +45°±5.7°with respect to the polarization direction of incoming illuminationlight.
 10. The structured illumination microscope apparatus according toclaim 2, wherein the direction of a fast axis of the second quarter waveplate is −45°±5.7° with respect to a ruling direction of the beamsplitter.
 11. The structured illumination microscope apparatus accordingto claim 2, further comprising a polarizer having a transmissionpolarization direction which is parallel with a ruling direction of thebeam splitter, immediately after the second quarter wave plate.
 12. Thestructured illumination microscope apparatus according to claim 2,wherein the phase difference between an ordinary beam and anextraordinary beam after passing through the first quarter wave plateand the phase difference between an ordinary beam and an extraordinarybeam after passing through the second quarter wave plate are within90°±5°, respectively.
 13. The structured illumination microscopeapparatus according to claim 2, wherein the relationship of 0.20φ1×sin(6θ1)≦φ2≦0.30φ1×sin (6θ1) is established, where θ1 is a first structuredillumination direction, φ1 is a difference of the phase differencegenerated by the first quarter wave plate from 90° (quarter wave), andφ2 is a difference of the phase difference generated by the secondquarter wave plate from 90° (quarter wave).
 14. An image formingapparatus comprising, light source means for producing linearlypolarized light, light splitting means, placed in a position that isoptically conjugate to a specimen, for splitting light from the lightsource means, a periodic structured light means for providing a periodicstructured light on a specimen by interference of the split light,shifting means for shifting the phase of the periodic structured light,producing means for producing images of light from the specimen onto animage plane, detecting means for detecting and recording the images ofthe specimen in the image plane, and processing means for producing areconstruction of the specimen by processing the recorded images whichare taken with different phase of the periodic structured light, whereina first quarter wave retarding means is located between the light sourcemeans and the light splitting means, the first quarter wave retardingmeans is mounted in a fixed orientation relative to the polarization ofthe light source, to produce circularly polarized light, and a secondquarter wave retarding means is located between the first quarter waveretarding means and the light splitting means, the second quarter waveretarding means is enabled to co-rotate with the light splitting means.15. An image forming apparatus comprising, light source means forproducing linearly polarized light, light splitting means placed in aposition that is optically conjugate to a specimen, to split the lightfrom the light source means into a plurality of beams of light, lightselecting means to select a subset of beams from among the plurality ofbeams of light, a periodic structured light means for providing aperiodic structured light on the specimen by interference of the splitlight, producing means for producing images of light from the specimenonto an image plane, detecting means for detecting and recording theimages of the specimen in the image plane, and processing means forproducing a reconstruction of the specimen by processing the recordedimages which are taken with different phase of the periodic structuredlight, wherein a first quarter wave retarding means is located betweenthe light source means and the light splitting means, the first quarterwave retarding means is mounted in a fixed orientation relative to thepolarization of the light source, to produce circularly polarized light,and a second quarter wave retarding means is located between the firstquarter wave retarding means and the specimen, the second quarter waveretarding means is enabled to co-rotate with the light selecting means.16. An illumination optical system which includes a beam splitterrotatably disposed near a conjugate position of a sample and splitting abeam from a light source into a plurality of beams, and which irradiatesillumination light that has been spatially modulated by allowing theplurality of beams to interfere near a sample surface, comprising: afirst quarter wave plate which is disposed between the light source andthe beam splitter, and of which direction of a fast axis is relativelyfixed with respect to a polarization direction of the light from thelight source; and a second quarter wave plate which is disposed betweenthe first quarter wave plate and the beam splitter, and is rotatablecoaxially with the rotation axis of the beam splitter with the samerotation angle as that of the beam splitter.
 17. An illumination opticalsystem which includes a beam splitter disposed near a conjugate positionof a sample, and splitting a beam from a light source into a pluralityof groups of beams, of which splitting directions are different, aroundan optical axis; and a beam selector for selecting and transmitting onlyone arbitrary group of beams out of the plurality of groups of beams,and which irradiates illumination light that has been spatiallymodulated by allowing the plurality of beams included in the one groupof beams to interfere near a sample surface, comprising: a first quarterwave plate which is disposed between the light source and the beamsplitter, and of which direction of a fast axis is relatively fixed withrespect to a polarization direction of the light from the light source;and a second quarter wave plate which is disposed between the beamsplitter and the beam selector, and of which rotation angle from theoptical axis is relatively fixed with respect to the beam selector.